Extended Data Fig. 8: Validations of Hop_DP2 binding to GR and of the in vivo importance of the client-binding pocket in Hop_DP2.

a–d, Using photoreactive, site-directed cross-linking to validate the Hop_DP2:GR interaction (a, the pink box) and (b, left). In a, Hop_DP2 is loosely packed and uses surface-exposed hydrophobic residues, shown in sticks, to interact with Hsp90A_amphi-α (shown in transparent surface and with hydrophobic residues in sticks) and Hsp90B^W320,F349. Suggested by modelling of the photoreactive cross-linker p-benzoyl-l-phenylalanine (pBpa) on various positions of Hop_DP2 to search for the position which is at the closest proximity to the GR_Helix1 density (b, right), the pBpa was placed at Hop^Q512 (b, left). The blue arrow on the right panel in b points at the selected position, Q512 (right panel, b). A time course of UV-exposed GR-loading complex analysed by SDS–PAGE and visualized by Coomassie staining (c). Whole fractions of GR-loading complex eluted from the size-exclusion column were exposed to UV using a gel imager (see also Methods). In c, arrows at 0 and 60 min indicate a reduced intensity of the GR band over the time course. Western blot of the SDS–PAGE gel after a 60 min UV exposure, using anti-MBP antibody to detect the MBP-tagged GR is shown in (d). Data in (c, d) are from one experiment. e–h, Hop_DP2’s client-binding/transfer function is crucial for cellular functions and client maturation. Hop^{L508 (L553 in Sti1)} is located on the hydrophobic palm (Fig. 2d) of Hop^DP2, interacting closely with the LXXLL motif of GR_{Helix 1} through hydrophobic interactions (e, left, middle and right). Mutations of Hop^L508 completely abrogated GR function in vivo (Sti1^L553A in Schmid et al. 2012³³), lead to growth defects (g), and failed to promote v-src maturation (h). The mutant Sti1^L553D accumulates at levels similar to WT Sti1 (f); data in f are from two independent experiments (see also Supplementary Fig. 10 for the uncropped gels/blots). Extracts from WT cells (JJ762), sti1 cells (JJ623) or sti1 cells transformed with a plasmid that expresses WT Sti1 or Sti1^L553D were analysed by SDS–PAGE and immunoblotted with a polyclonal antisera specific for Sti1. Loading control is antibody against mitochondrial protein Tim44. In g, sti1-L553D is inviable in hsc82hsp82 cells expressing hsc82-G309S. hsc82hsp82 (JJ117) or sti1hsc82hsp82 (JJ1443) strains harbouring YEp24-HSP82 were transformed with plasmids expressing WT HSC82 or hsc82-G309S. Strains that lacked STI1 were also transformed with an empty plasmid or a plasmid expressing WT STI1 or sti1-L553D. Transformants were grown in the presence of 5-FOA for 3 days to counter-select for the YEp24-HSP82 plasmid. STI1 is essential under these conditions and the growth of cells expressing sti1-L553D was indistinguishable from those expressing the empty plasmid. In h, WT cells, sti1 cells or sti1 cells transformed with a plasmid that expresses WT Sti1 or Sti1-L553D were transformed with an empty plasmid or a plasmid that expresses GAL-v-src. v-src induction in the presence of galactose sharply reduces the growth of WT cells, but not cells lacking STI1. The growth of cells expressing sti1-L553D was very similar to those expressing the empty plasmid, indicating that sti1-L553D is unable to support v-src function. The growth of cells in the presence of glucose was indistinguishable. 10-fold serial dilutions of cultures were grown for 3 days in the presence of galactose or glucose.